The protozoan parasite Toxoplasma gondii is an important opportunistic infection causing life threatening disease in patients with HIV-AIDS and other immune compromising conditions. The high seroprevalence of the T. gondii worldwide but low incidence of symptomatic disease reflects the fact that the acute phase of infection is effectively handled by the immune response. Unfortunately a sterile cure is not achieved. Rather the parasite establishes a life-long chronic infections mediated by tissue cysts typically formed in the brain and muscle. The tissue cyst is protected by a heavily glycosylated cyst wall. Despite the importance of glycosylation, little is known about these pathways in the parasite. With this work we address the contribution of N-linked glycosylation in Toxoplasma. We propose to use lectin affinity coupled with mass spectrometric identification to catalogue the diversity of N-glycosylated glycoproteins in tissue cysts. We further plan to conditionally knock out a key gene in the N-glycosylation, TgALG7, in order to assess its role in parasite biology with a focus on protein targeting to its diverse unique secretory organelles. Given the central role for glycosylation in the tissue cyst we propose an experimental strategy to selectively knock out gene expression in the tissue cyst. This technology will be applied to establishing the contribution of TgALG7 and by extension N-glycosylation to tissue cyst formation and stability. This work represents the first in depth study of the cell biologic basis of protein glycosylation i Toxoplasma using state of the art approaches to modulate gene expression. PUBLIC HEALTH RELEVANCE: Toxoplasma gondii causes serious illness in immune compromised individuals including patients with HIV- AIDS. Our understanding of the role of glycosylation in parasite biology is very limited. With this work we directly explore N-glycosylation in the with a focus the dormant tissue cyst form. We hope to establish a strong foundation for future studies in this under explored area of investigation.